Page 173 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

SDIRSACR Oncology Insights

Annexin V-FITC and propidium iodide staining.
Results: Expression of PKM2, CTSB, and CTSD was significantly higher in malignant melanoma than in normal skin or
benign nevi. Both SH and CQ reduced A375 cell viability in a dose-dependent manner, with their combination exhibiting
enhanced cytotoxicity. The calculated α value was greater than 1, indicating a synergistic interaction between SH and
CQ. Annexin V/PI flow cytometry revealed that both agents induced apoptosis, which was significantly amplified by the
combined treatment.
Conclusions: Dual targeting of melanoma-upregulated PKM2 with SH and cathepsins B/C with CQ resulted in synergistic
proapoptotic effects in A375 melanoma cells, suggesting that this combined approach may represent an effective
therapeutic strategy for melanoma.

Acknowledgments and funding: This research was funded by the Ministry of Science, Technological Development and
Innovation of the Republic of Serbia, grant numbers 451-03-137/2025-03/ 200110 and 451-03-136/2025-03/200007.
Lj.H.T. has received a research donation from the nonprofit organization Climbers Against Cancer.

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Mechanistic insights into synergistic antimelanoma activity of glycolysis inhibition and lysosomal
destabilization

Milica Kosić , Mihajlo Bošnjak , Miloš Mandić , Ljubica Vučićević , Maja Misirkić Marjanović , Verica Paunović ,
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Danijela Stevanović , Kristina Janjetović , Aleksandar Paunić , Aleksandra Aničin , Vladimir Trajković , Ljubica Harhaji
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Trajković 2
1Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia
2Institute for Biological Research “Siniša Stanković” – National Institute of the Republic of Serbia, University of Belgrade, Belgrade,
Serbia
Keywords: melanoma, energy metabolism, glycolysis, lysosomes, cathepsins
Background: Melanoma cells exhibit high glycolytic activity and increased lysosomal fragility. Cathepsins, overexpressed
in melanoma, support tumor progression but trigger cell death upon lysosomal membrane permeabilization (LMP).
This study investigated the mechanisms by which the glycolytic inhibitor 2-deoxy-D-glucose (2DG) synergized with LMP
inducer L-leucyl-L-leucine methyl ester (LLOMe), to induce death of human A375 melanoma cells.
Materials and Methods: Cell viability was measured by the crystal violet test. Lysosomal integrity was evaluated using
acidophilic dyes LysoTracker and acridine orange via fluorescent microscopy and flow cytometry. Fluorometric assays
were used to measure mitochondrial membrane potential (JC-1), mitochondrial superoxide production (MitoSOX™
Red), glycolytic activity (pH-Xtra™ Glycolysis Assay), oxidative phosphorylation (MitoXpress® Xtra Oxygen Consumption
Assay), and intracellular ATP levels (ATP assay kit). Cells were pretreated with cysteine cathepsin inhibitors (E64d,
MG132), aspartic cathepsin inhibitor (Pepstatin A), lysosomal acidification inhibitor (bafilomycin A1), antioxidant NAC,
and energy booster L-carnitine to further dissect the contribution of specific pathways.
Results: LLOMe synergized with 2DG to induce cytotoxicity in A375 cells. LLOMe, with or without 2DG, induced LMP,
evidenced by reduced LysoTracker and acridine orange signals. Bafilomycin A1 rescued cell viability, likely by preventing
LLOMe accumulation and activation within lysosomes. E64d and MG132, but not Pepstatin A, partially restored
viability, implicating cysteine cathepsins in the observed toxicity. LLOMe, with or without 2DG, triggered mitochondrial
depolarization and mitochondrial superoxide production, which was prevented by bafilomycin A1 and MG132. NAC
rescued cells from 2DG+LLOMe-induced cytotoxicity. Metabolic assays showed that 2DG reduced glycolysis and LLOMe
suppressed oxidative phosphorylation, leading to synergistic ATP depletion, while L-carnitine protected cells from the
combined treatment.
Conclusions: LLOMe-induced LMP and cysteine cathepsin release lead to mitochondrial dysfunction and OXPHOS
inhibition, which, together with 2DG-mediated glycolysis blockade, result in energy collapse and cell death, highlighting
the therapeutic potential of dual glycolytic–lysosomal targeting.

Acknowledgments and funding: This work was funded by the Ministry of Science, Technological Development and
Innovation of the Republic of Serbia (Contract No. 451-03-137/2025-03/ 200110 and 451-03-136/2025-03/200007)
and the non-profit international charity “Climbers Against Cancer” (donation number #3).

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